The VICTORIA study (ClinicalTrials.gov, Identifier: NCT06313645) is a prospective single-center cohort study involving patients suffering from chest pain undergoing coronary angiography and revascularization procedures at the Cardiology Department of Niguarda Hospital, Italy. The study adheres to the general principles outlined in the Declaration of Helsinki [30] and the rules of good clinical practice (GCP).

A total of 300 consecutive patients with acute coronary syndrome (ACS) will be enrolled, including those presenting with myocardial ischaemia at rest or on minimal exertion in the absence of acute cardiomyocyte injury/necrosis (UA), NSTEMI, and patients with acute STEMI, as defined by the European Society of Cardiology [31].

The study will also recruit patients with documented chronic coronary artery disease admitted for coronary angiography or elective revascularization (stable angina) and patients with positive stress test but with non-angiographically significant coronary artery disease. Exclusion criteria will include patients in cardiogenic shock, with congestive heart failure, valve disease, acute renal failure, undergoing coronary artery bypass grafting, or with active cancer.

Clinical characteristics and signs for ACS diagnosis are described in Table 1.

Patients will undergo follow-up within 12 months, either through hospital records or via in-person or telephone interviews, to evaluate occurrences of all-cause mortality and major adverse cardiovascular events (MACE).

Prior to enrollment, all eligible participants will need to provide informed written consent. The study will document all clinical characteristics for each patient, comprising demographics, complete medical history, medication details, and previous diagnostic and therapeutic interventions. Upon admission, patient laboratory data, encompassing total blood count and standard biochemistry parameters, will be collected. Experienced interventional cardiologists will gather angiographic parameters, including lesion characteristics and coronary dominance.

Additionally, specific details concerning plaque characteristics such as thickness, type of fibrotic cap, presence of intraplaque hemorrhage, and inflammation, will be obtained from patients undergoing evaluation of plaque morphology and composition using intravascular ultrasound (IVUS) or optical coherence tomography (OCT).

Within 24 h of admission, an echocardiographic assessment will be performed. This assessment will involve quantification of cardiac chamber sizing, measurements of the left ventricle (LV), and evaluation of conventional LV diastolic parameters using Doppler measurements, all in accordance with current published criteria [32, 33].

Before the angiography procedure, roughly 10 mL of peripheral blood will be drawn from each consenting participant in the catheterization laboratory for the collection, preparation, and storage of different biosamples. Aliquots of samples will be promptly frozen following standardized procedures. The study design is illustrated in Figure 1.

The biomaterials utilized for molecular analysis will comprise serum, plasma (EDTA, citrate), genomic DNA (gDNA), RNA from whole blood, and peripheral blood mononuclear cells (PBMCs). Specifically, we will assess biomarkers of cellular aging (TL and mtDNA content) and the dysregulation of specific non-coding RNAs (lncRNA TERRA, MitomiR). Moreover, the project will investigate the potential role of circulating extracellular vesicles (EVs) in modulating the telomere-mitochondria link through miRNA-sensitive mechanisms. To gain insight into cellular effects and communication, we will also evaluate levels of secreted pro-inflammatory cytokines and EV cargo in the supernatant of the whole blood culture. Figure 2 provides an overview of the experimental workflow and planned measurements. After the collection process, the biological samples will undergo analysis using state-of-the-art methods to determine the levels of selected biomarkers.

TL and mtDNAcn will be assessed using DNA extracted from 200 µL of blood samples via quantitative real-time PCR, following standardized protocols. Briefly, TL will be determined by measuring the ratio of telomere repeat copy number (T) to a single-copy gene (S) and calculating the T/S ratio. The relative TL will be calculated using the formula “T/S ratio = 2^(–ΔΔCt)”, where ΔCt = Ct telomere – Ct single-copy gene. The T/S ratio provides an average TL across all leukocytes. For the quantification of mtDNAcn, the NDI1 gene in the undeleted region of the mtDNA reference sequence (mtNDI1) will serve as an internal control, while the human ß-globin gene from gDNA will be amplified by PCR in both gDNA and mtDNA. mtDNA-CN will be determined using the (2^ΔCt) method, where ΔCt = Ct mtNDI1 – Ct gDNA.

A panel of MitomiRs known to be linked to senescence (miR-21-5p, miR-146a-5p, miR-181a-5p, miR-210, and miR-34a) and the expression levels of TERRA lncRNAs will be measured following RNA extraction from blood. Initially, total RNA (1 µg) will be reverse transcribed into cDNA using the TaqMan miRNA Reverse Transcription Kit (Thermo Fisher Scientific, USA). Subsequently, RT-qPCR will be performed utilizing sequence-specific TaqMan MicroRNA Assays (Thermo Fisher Scientific, USA) and TaqMan Universal PCR Master Mix (without AmpErase^® UNG). Specific oligonucleotides will be used to amplify TERRA transcripts from different chromosome ends. Relative transcript levels will be calculated using the ΔΔCT method and normalized to the reference genes.

Exosomes will be isolated from serum using an Exosome Isolation Kit (Invitrogen, Life Technologies) according to the manufacturer’s instructions. The quality, concentration, and size of exosomes will be assessed by nanoparticle tracking analysis (NTA). RNA extraction from exosomes will be performed using a specific miRNA Kit (Qiagen, Hilden, Germany). Library preparation, quality control, and next-generation sequencing procedures will be outsourced to a specialized company. Finally, the expression of the selected dysregulated miRNAs will be confirmed by qRT-PCR analysis.

Pro-inflammatory cytokines and chemokines [interleukins (IL)-2, IL-6, IL-8, IL-18, tumor necrosis factor α (TNFα), monocyte chemoattractant protein-1 (MCP-1)] will be measured in patient plasma or serum using commercially available Human ELISA Kits (FineTest, Wuhan, Hubei, China). Briefly, the microplates will be coated with monoclonal antibodies specific to cytokines. Standards and supernatants will be added to the wells of the microplate and run in duplicate. Cytokine concentrations (pg/mL) will be determined against a standard concentration curve. Additionally, the expression levels of cell cycle inhibitor expression (p16 and p21) will be quantified by RT-qPCR analysis in isolated PBMCs (p16 and p21) will be also performed by RT-qPCR analysis in isolated PBMCs.

To detect a medium effect size (f = 0.25) in the difference of the mean LTL value between the groups, we estimate that a sample size of at least 232 total patients is required, with an alpha level of 0.05 and a power of 90% or higher. Accounting for a at least 10% dropout rate, the total number of patients to be enrolled should be approximately 300.

In terms of statistical tests, normal distribution of data will be assessed using the Kolmogorov-Smirnov test. Continuous variables will be presented as mean, standard deviation, median, first, and third quartiles. Categorical variables will be expressed as numbers and percentages.

Comparisons between two groups will be conducted using Student’s t-test for independent samples for continuous variables, and the chi-square test or Fisher’s exact test for categorical variables. Comparisons between more than two groups will be tested with one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc tests for pairwise comparisons. If the assumption of normal data distribution is not met, equivalent non-parametric tests will be employed.

Pearson’s or Spearman’s coefficient will be calculated to investigate univariate correlations between variables. Event-free survival curves will be constructed using the Kaplan-Meier model and tested with the log-rank test between different groups. The Cox proportional hazards model, both univariate and multivariate, will be used to identify the predictive value of each variable vs. MACE events; data will be expressed with hazard ratios and their 95% confidence intervals.

Data management and statistical analyses will be conducted using SPSS software, version 26 (IBM SPSS Statistics) and StatView statistical package, version 5.0.1 (Abacus Concepts, Berkeley, CA, USA).